von Willebrand factor (VWF) is a multi-domain plasma protein secreted by endothelial cells. In hemostasis, VWF binds and crosslinks platelets to one another and the vessel wall to form the platelet plug. VWF also binds to and stabilizes factor VIII (FVIII) in the coagulation cascade. VWF mutations cause the most common heritable bleeding disorders called von Willebrand disease (VWD). The D1, D2, and DD3 assemblies in VWF are specialized domains that enable biosynthesis of VWF into ultralong concatemers that are stored as helical tubules in Weibel-Palade bodies (WPBs). DD3 also binds FVIII. Long length enables VWF to sense flow. Changes in flow at sites of bleeding activate VWF by 1) elongating coiled VWF concatemers into a thread-like conformation that exposes previously buried A1 domains and 2) activating a high-affinity state of VWF A1 domains that bind platelet glycoprotein Ib? (GPIb?) for platelet plug formation. High-resolution structures of D assemblies and the high-affinity state of A1 are lacking. In Aim 1, we will determine the structure of the high- affinity state of A1. Unfolding studies show that VWF A2 and A3 domains have two states, whereas A1 has three: native, intermediate, and unfolded. Preliminary studies show that truncating the O-glycosylated linkers N- and C-terminal destabilizes the native state of A1 and increases affinity for GPIb?. We propose that the intermediate state corresponds to the high-affinity state of A1. We test the hypothesis that further truncation of the linkers flanking A1, gain-of-function mutations (e.g. activating VWD mutations), and the allosteric activator ristocetin all increase A1 affinity for GPIb? by stabilizing the intermediate state over the native state. We will use combinations of truncations, mutations, and ristocetin to stabilize A1 in the intermediate state and to determine the crystal structure of the putative high-affinity state of A1 and its complex with GPIb?. Aim 2 will determine structures of DD3 and the DD3 dimer. Our preliminary crystal structure of the DD3 monomer shows how the C8, TIL, and E modules pack around the VWD module to form the D3 assembly. D protrudes from the D3 assembly. The two cysteines that have been proposed to form the inter-dimer disulfide bonds are buried. We will solve the structure of a DD3 dimer (DD3)2 or a D3 dimer with the protruding D removed to define the structural rearrangements required for DD3 dimerization. Proposed disulfide rearrangement that precedes dimerization will be verified by mutation and in vitro reconstitution. As backup, we will pursue a cryo- EM structure of VWF helical tubules to determine the structure of (DD3)2 and how D assemblies enable formation of highly ordered tubules. Aim 3 uses crystallography to understand how D?D3 binds FVIII, which has the potential through protein engineering to revolutionize replacement FVIII therapy in hemophilia A. As an alternative strategy, we will determine a cryoEM structure of a D?D3 complex with FVIII. Better structural understanding of VWF D assemblies and the high-affinity state of A1 has important therapeutic implications for stroke, thrombosis, VWD, and hemophilia A.